The present invention relates to a purified xcex2-galactosidase specific for lactose.
xcex2-galactosidase catalyzes the hydrolysis of lactose disaccharide into its constituent monosaccharides, glucose and galactose.
This enzyme is widely distributed in numerous micro-organisms, plant and animal tissues.
The ability of xcex2-galactosidase to hydrolyse lactose into galactose is applied in food industry, particularly in the field of dairy products because of the nutritional (lactose intolerance), technological (crystallisation) and environmental (pollution) problems associated with lactose (Triveni P. S., 1975, CRC Critical Reviews in Food Technology 325-354). The added value gained by the hydrolysis of lactose, to its constituent monosaccharides glucose and galactose, lies in the increased usefulness of hydrolysed lactose as a food carbohydrate. Lactose itself has limited use in this respect because of its relatively low sweetness, solubility and digestibility, but the hydrolysis products of lactose, i.e. glucose and galactose, are superior in all of these respects. Increased sweetness and solubility improve the technical usefulness of whey products while the increased digestibility of hydrolysed lactose also offers the opportunity of supplying milk solids to populations which have hitherto been unable to consume milk products because of their inability to hydrolyse lactose in the digestive tract.
The xcex2-galactosidase can be applied to the production of low-lactose milk and in the production of galactose or glucose from lactose contained in milk serum which is formed in large amount in the process of producing cheese.
The major applications for lactose hydrolysis are listed below.
a) Liquid milk. Lactose hydrolysis in liquid milk improves digestibility for lactose intolerant consumers. In flavoured milks, lactose hydrolysis increases sweetness and enhances flavours.
b) Milk powders. Lactose hydrolysed milk powders for dietetic uses, especially for infants with temporary xcex2-galactosidase deficiency.
c) Fermented milk products. In some cases, lactose hydrolysis in milk used for the manufacture of cheese and yoghurt can increase the rate of acid development and thus reduce processing time.
d) Concentrated milk products. Lactose hydrolysis in concentrated milk products (e.g. sweetened condensed milk, ice cream) prevents crystallisation of lactose.
e) Whey for animal feed. Lactose hydrolysis in whey enables more whey solids to be fed to pigs and cattle and also prevents crystallisation in whey concentrate.
f) Whey. Lactose hydrolysed whey is concentrated to produce a syrup containing 70-75 per cent solids. This syrup provides a source of functional whey protein and sweet carbohydrate and is used as a food ingredient in ice cream, bakery and confectionery products.
The conventional approach in food processing is to carry out the hydrolysis of lactose at 40xc2x0 C. during approximately four hours. (T. Godfrey and J. Reichelt in : xe2x80x9cIndustrial Enzymology: the application of enzymes in industryxe2x80x9d; The Nature Press, Mac Millan Publishers Ltd, GB, 1983). However, milk or lactose solution as a raw material is a preferable nutrition source for bacteria. As the result, the putrefaction owing to the saprophyte contamination during the treatment is a serious problem in the food production. Thus, the fact is that the conventional xcex2-galactosidase is not put into practical use.
Attempts to solve these problems consisted in using thermophilic enzymes as described in U.S. Pat. No. 4,237,230 and U.S. Pat. No. 4,007,283 but a problem of high energetic cost still remains.
On another hand, cold-adapted xcex2-galactosidases have been studied (Trimbur D. E. and al., 1994, Appl. Environ. Microbiol. 60:4544-4552; Rahim K. A. A. and Leb B. H., 1991, Biotechnol and Appl. Biochem., 13, 246-256).
However, these xcex2-galactosidases generally known in the prior art, when used for food processing, all have one or more disadvantages such as low enzyme activity and low stability at a temperature below 20xc2x0 C., narrow range of optimum pH and the inhibition of enzymatic action by a reaction product, such galactose or others products particularly calcium.
The object of the present invention is to hydrolyse lactose by using a xcex2-galactosidase, which could overcome the above-mentioned drawbacks which are usually associated to this process, while advantageously avoiding contamination problems during the hydrolysis process and lowering the energy consumption.
This problem is solved according to the present invention by a purified cold-active xcex2-galactosidase, specific for lactose, having a stable enzymatic activity at temperatures up to below 8xc2x0 C., preferably up to below 6xc2x0 C., and specifically at 4xc2x0 C., which corresponds to refrigerating conservation temperature for dairy products. This enzyme of the invention is consequently able to hydrolyse lactose in dairy products and stable enzymatic activity at temperatures up to below 8xc2x0 C., preferably up to below 6xc2x0 C., and specifically at 4xc2x0 C., which corresponds to refrigerating conservation temperature for dairy products. This enzyme of the invention is consequently able to hydrolyse lactose in dairy products and milk processing at such a low temperature that saprophytes are hindered to proliferate. The hydrolysis of lactose can be carried out in these refrigeration conditions with no need of a particular treatment to the dairy product concerned.
According to the invention, an enzymatic activity is considered as stable when, in the concerned conditions, the enzyme is capable of lasting long enough to obtain the desired effect, for example, the hydrolysis of a substrate.
According to an embodiment of the invention, the cold-active xcex2-galactosidase has a stable enzymatic activity between 0 and 50xc2x0 C.
Advantageously, the cold-active xcex2-galactosidase according to the invention has a stable enzymatic activity at a pH range from 6 to 10, preferably from 6 to 8.
Preferably, the cold-active xcex2-galactosidase according to the invention has a stable enzymatic activity in presence of calcium and/or galactose, meaning that the activity of this enzyme is neither inhibited by its reaction product nor by products being present in milk. This property allows to use efficiently this enzyme in milk treatment.
Such a cold-adapted xcex2-galactosidase according to the invention attains the level of practical application, having simultaneously the following properties:
(1) Having a sufficient stability in the neighbourhood of 0 to 10xc2x0 C.
(2) Having a sufficient enzymatic activity at a pH range from 6 to 10
(3) Having an enzymatic activity non inhibited by reaction products or other products substantially present in milk, such calcium.
According to an advantageous embodiment of the invention, the enzyme can be inactivated at a pasteurisation temperature. This property of the enzyme according to the present invention allows to apply the xcex2-galactosidase according to the invention and to stop the enzymatic reaction of lactose hydrolysis without any additional step during a current milk treatment.
Another object of the present invention is a strain of an isolated psychrophilic bacterium capable of producing a cold-active xcex2-galactosidase according to the present invention. A preferable strain is Pseudoalteromonas haloplanktis deposited on the Nov. 4, 1999, under the Budapest Treaty at the Belgian Coordinated Collections of Microorganisms (BCCM(trademark)), Laboratorium voor Microbiologiexe2x80x94Bacterixc3xanverzameling (BCCM(trademark)/LMG), Universiteit Gent, K. L. Ledeganckstraat 35, 9000 Gent, Belgium, with the Accession NO LMG P-19143 and variants and mutants derived therefrom.
To purify a cold-active xcex2-galactosidase according to the invention, a bacterium living in the Antarctic area was isolated and characterised in order to study how its enzymes, and particularly, the xcex2-galactosidase was adapted to cold. These studies led to the purification of the xcex2-galactosidase, meaning that this protein was obtained substantially free of other proteins as determined by Sodium Dodecyl Sulphate Polyacrylamide gel Electrophoresis (SDS-PAGE) using protein purification steps known in the art.
Micro-organisms can be divided in categories depending on the temperature at which they can proliferate. The widely accepted definition by Morita (Psychrophilic bacteria. Bacteriol. Rev. 39: 144-167; 1975.) proposes that psychrophiles include organisms having optimum growth temperatures  less than 15xc2x0 C. and upper cardinal temperatures around 20xc2x0 C., although they are able to multiply and to carry out all their biochemical functions near the normal freezing point of water. The mesophilic bacteria proliferate at an average temperature range between 25 and around 40xc2x0 C. Thermophilic micro-organisms proliferate at a temperature above 50xc2x0 C. and hyperthermophilic micro-organisms grow at temperatures above 80xc2x0 C.
As a general rule, micro-organisms which are pathogenic for human and animals are mesophilic, so it is interesting to carry out industrial food processing at low temperatures to avoid the possible proliferation of such pathogens.
It is still an object of the present invention to provide a DNA sequence comprising a gene which encodes a polypeptide having the biological activity of the cold-active xcex2-galactosidase according to the invention. A preferable DNA sequence is shown in SEQ ID NO: 1 and a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 is preferable.
Another object of the invention is a recombinant plasmid suited for transformation of a host, capable of directing the expression of a DNA sequence according to the invention in such a manner that the host expresses said polypeptide having the biological activity of the cold-active xcex2-galactosidase in recoverable form. According to the invention another object is the so transformed host.
A variety of host-expression systems may be conceived to express the cold-active xcex2-galactosidase coding sequence, for example bacteria, yeast, insect cells, plant cells, mammalian cells, etc.
Particularly, in yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review see Grant and al., 1987, Expression and secretion vectors for yeast, in Methods in Enzymology, Eds. Wu and Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp. 516-544.
It is also an object of the present invention to provide a process for purifying the cold-active xcex2-galactosidase according to the invention from a psychrophilic bacterium as well as to provide a process for producing cold-active xcex2-galactosidase according to the invention in a transformed host.
These and other objects of the present invention will be apparent from the following disclosure.
Other characteristics of the present invention are listed in the annexed claims.